The present invention relates to liquid chromatography packing materials, and more particularly it relates to an improved reverse phase packing material for use in blood serum analysis and to a method for making and using such a packing material.
The use of liquid chromatography as a means for blood serum analysis has become quite popular. Liquid chromatography using a reverse phase packing material has been found to be an effective tool in both qualitative and quantitative analysis for drug substances in blood, serum, or plasma. Typically the reverse phase packing material is made up of bonded alkyl silica and most typically the packing is a porous silica having an octadecylsilane (ODS) bonded to it.
Work has recently been done on improving the efficiency of ODS bonded silica packings. See, for example, Marshall et al, "Synthesis of L C Reversed Phases of Higher Efficiency by Initial Partial Deactivation of the Silica Surface," Journal of Chromatography Science, Vol. 22., June 1984, pp. 217-220, where it is suggested that pretreating silica with a small amount of end-capping reagent (such as trimethylchlorosilane), followed by exhaustive octadecylation, yields reverse phase packings of higher efficiency.
Although the efficiency of such packing materials is good, they have a limited life. While ODS packings absorb drug substances from the sample, they also absorb proteinaceous substances which tend to interfere with fractionation of the drug substance from other solutes contained in the sample. This eventually leads to a complete fouling of the chromatographic column. Therefore, it has previously been necessary to carry out a preliminary sample preparation procedure to remove the troublesome proteins.
In the most conventional way, the proteins are precipitated, the aqueous supernatant is extracted with a water-immiscible organic solvent, the organic solvent is removed from the extract by evaporation, and the analyte residue is reconstituted in mobile phase before analysis by high-pressure liquid chromatography (HPLC). This method is very time-consuming and cost-inefficient.
A second method currently employed involves the adsorption of analytes onto a reverse phase packing of octadecylsilane bonded to silica in a small disposable column. Although this technique can be automated, the columns can be used for only one sample because proteins remain on the packing, and as a result the technique is also cost-inefficient for multiple samples.
In a third method, a reverse phase packing of octadecylsilane bonded to silica is introduced into a precolumn, which is separated from, but connectable to, an analytical column by a switching valve arrangement. Serum samples are injected directly into the precolumn, where the proteins are denatured and accumulated, and the deproteinated analyte solution is passed into the analytical column for fractionation. After approximately three injections, the precolumn must be backflushed to remove the protein residue. This interruptive backflush is time-inefficient for a large number of samples. Furthermore, the octadecylsilane packing eventually deteriorates because proteins cannot be completely removed therefrom.
The three above-mentioned techniques are discussed in Pinkerton et al U.S. Pat. No. 4,544,485. Pinkerton et al discloses an improvement in the form of "internal surface reversed phase" (ISRP) packing materials. The Pinkerton ISRP packings, and variations thereof, are described not only in Pinkerton U.S. Pat. No. 4,544,485, but also Hagestam, I. and Pinkerton, T., "Internal Surface Reversed-Phase Silica Supports for Liquid Chromatography", Analytical Chemistry, Vol. 57, 1985, pp. 1757+; Szczerba et al, "HPLC Column Finds Drugs in Serum", Research & Development, September 1986, pp. 84-86; and Hagestam, I. and Pinkerton, T., "Internal Surface Reversed-Phase Silica Support Prepared with Chymotrypsin", Journal of Chromatography, Vol. 351, 1986, pp. 239-248.
Basically, the Pinkerton patent and publications state that the Pinkerton ISRP packing materials allow for the direct injection of serum samples into the chromatographic column, thus eliminating time-consuming preparation required to rid samples of the proteins that can clog pores and interfere with the quality of separations in conventional ODS reverse phase liquid chromatography. The Pinkerton ISRP packing material are described as having a hydrophobic partitioning phase present only on the internal surfaces of the porous silica support, while a hydrophilic phase, which is non-adsorptive to proteins, covers the external surfaces. Serum proteins, being too large to enter into the pores of the support, "see" only the non-adsorptive hydrophilic external phase and elute rapidly in the interstitial void volume giving the column a larger useful life. The small hydrophobic analytes, on the other hand, can penetrate into the porous interior of the support and interact with the hydrophobic partitioning phase.
Generally, the Pinkerton ISRP packing materials are prepared by taking porous silica having glycerolpropyl groups bonded thereto, covalently bonding a hydrophobic polypeptide to some fraction of the glycerolpropyl groups, and then treating the bonded silica with an enzyme which cleaves the hydrophobic polypeptide from the external surface but not the internal surface. Two methods for producing the Pinkerton ISRP packing materials have been described in the above-mentioned literature. The first involves binding a hydrophobic glycyl-L-phenylalanyl-L-phenylalanine (Gly-L-phe-L-phe) tripeptide to the surface of glycerolpropyl bonded porous silica particles. This is followed by enzymatic cleavage of the phenylalanine moieties on the external surface via carboxypeptidase A. In the second method, a butoxy-L-phenylalanine (boc-L-phe) partitioning phase is bound to an alkylamine bonded silica support. The enzyme chrymotrypsin is then utilized to cleave the boc-L-phe phase from the external surface, and the remaining external residual alkylamine groups are capped with glycidol.
However, even with the Pinkerton ISRP packing materials it has been found desirable to use a "guard column", i.e. a small disposable cartridge packed with spherical ISRP materials, to prolong the ISRP column life. See, for example, page 86 of the Szczerba et al article mentioned above.
Accordingly, while the concept of a dual internal surface/external surface silica packing, such as the Pinkerton ISRP packing materials, is a good one, improved packing materials having an even longer life would be desirable.